All molybdenum-containing enzymes examined so far, except nitrogenase, contain an organic moiety as an essential component of a common molybdenum cofactor. In our laboratory we have characterized this molecule as a novel sulfur-containing pterin and have obtained evidence for the presence of a labile complex between the pterin, now called molybdopterin, and Mo. The fact that all but one of the molybdoenzymes tested gave rise to fluorescent derivatives similar to those obtained from the Mo cofactor of chicken liver sulfite oxidase and bovine milk xanthine oxidase attests to the presence of a pterin as a component of the Mo cofactor in all these enzymes. We have also shown that pleiotropic mutants, lacking all Mo enzyme activities, in E. coli, N. Crassa, Drosophila and human lack molybdopterin. The future goals of our laboratory include 1) elucidation of the pathway of biosynthesis of molybdopterin in E. coli and other systems, including the characterization of the enzymes and precursors involved in the pathway; 2) testing the possibility that molybdopterin could be a component of more than one form of coenzyme in diverse enzymes; 3) determination of the oxidation state of the pterin ring in the Mo cofactors of several purified enzymes, and examining whether alteration of the oxidation state of the pterins would lead to loss of enzyme activity; and 4) cloning of the gene for sulfite oxidase in order to obtain the apoprotein needed for full characterization of the interaction between the Mo cofactor and an apoprotein and for probing the reason for the absence of sulfite oxidase protein in the tissues of molybdopterin-deficient patients. Attempts will also be continued for the isolation of a stable form of active Mo cofactor for structural elucidation by various techniques. We also propose to explore the mechanism whereby the Mo cofactor of xanthine dehydrogenase acquires a terminal sulfur ligand--a process which appears to be absent in the pleiotropic mal mutant of Drosophila.